Abstract

MUC4, a transmembrane mucin, is aberrantly expressed in pancreatic adenocarcinomas while remaining undetectable in the normal pancreas. Recent studies have shown that the expression of MUC4 is associated with the progression of pancreatic cancer and is inversely correlated with the prognosis of pancreatic cancer patients. In the present study, we have examined the phenotypic and molecular consequences of MUC4 silencing with an aim of establishing the mechanistic basis for its observed role in the pathogenesis of pancreatic cancer. The silencing of MUC4 expression was achieved by stable expression of a MUC4-specific short hairpin RNA in CD18/HPAF, a highly metastatic pancreatic adenocarcinoma cell line. A significant decrease in MUC4 expression was detected in MUC4-knockdown (CD18/HPAF-siMUC4) cells compared with the parental and scrambled short interfering RNA–transfected (CD18/HPAF-Scr) control cells by immunoblot analysis and immunofluorescence confocal microscopy. Consistent with our previous observation, inhibition of MUC4 expression restrained the pancreatic tumor cell growth and metastasis as shown in an orthotopic mouse model. Our in vitro studies revealed that MUC4-associated increase in tumor cell growth resulted from both the enhanced proliferation and reduced cell death. Furthermore, MUC4 expression was also associated with significantly increased invasiveness (P ≤ 0.05) and changes in actin organization. The presence of MUC4 on the cell surface was shown to interfere with the tumor cell-extracellular matrix interactions, in part, by inhibiting the integrin-mediated cell adhesion. An altered expression of growth- and metastasis-associated genes (LI-cadherin, CEACAM6, RAC1, AnnexinA1, thrombomodulin, epiregulin, S100A4, TP53, TP53BP, caspase-2, caspase-3, caspase-7, plakoglobin, and neuregulin-2) was also observed as a consequence of the silencing of MUC4. In conclusion, our study provides experimental evidence that supports the functional significance of MUC4 in pancreatic cancer progression and indicates a novel role for MUC4 in cancer cell signaling. (Mol Cancer Res 2007;5(4):309–20)

Keywords:

MUC4

Mucin

Pancreatic cancer

Proliferation

Survival

Introduction

Mucins are heavily glycosylated proteins that establish a selective molecular barrier at the epithelial surface and engage in signal transduction pathways that regulate morphogenesis (1, 2). In addition, mucins influence many cellular processes, including growth, differentiation, transformation, adhesion, invasion, and immune surveillance (1, 3). Previous studies have shown that MUC4, a transmembrane mucin, is expressed by epithelial cells in a variety of tissues (3-5). In the pancreas, however, MUC4 is not expressed under normal conditions, although its aberrant expression is reported in premalignant and malignant pancreatic lesions as well as in several pancreatic cancer cell lines (3, 6-10). Recent studies from our laboratory and elsewhere have indicated a strong association between MUC4 overexpression and pancreatic carcinogenesis, thereby implicating MUC4 as a novel target for diagnosis, prognosis, and therapy (3, 6-12).

MUC4 is a high-molecular-weight glycoprotein with multidomain organization (13-15). The deduced full-length amino acid sequence of the MUC4 apoprotein shows the presence of a leader peptide, a serine and threonine rich nontandem repeat region, central large tandem repeat domain containing 16-amino acid repetitive units, regions rich in potential N-glycosylation sites, two cysteine-rich domains, a putative GDPH proteolytic cleavage site, three epidermal growth factor–like domains, a hydrophobic transmembrane domain, and a short cytoplasmic tail. MUC4 mucin shares many structural similarities with the sialomucin complex (SMC/ratMuc4), which has previously been shown to facilitate tumor progression by multiple mechanisms (3, 13, 16-19). SMC/Muc4 is a heterodimeric glycoprotein composed of an O-glycosylated mucin subunit, ascites sialoglycoprotein-1, and an N-glycosylated transmembrane subunit, ascites sialoglycoprotein-2. Similarly, MUC4 also possesses two subunits: an extracellular mucin-like subunit, MUC4α, and a growth factor–like transmembrane subunit, MUC4β, containing three epidermal growth factor–like domains (3). The SMC/Muc4 is known to act as an intramembrane ligand for the receptor tyrosine kinase ErbB2/HER2/neu, inducing its limited phosphorylation via one of the epidermal growth factor domains (17, 20, 21). On the other hand, a recent study from our laboratory has indicated that MUC4 may modulate HER2 signaling by regulating its expression (11).

In the present study, we have elucidated the multiple roles of MUC4 in pancreatic cancer progression. The silencing of MUC4 was achieved by stable expression of a MUC4-specific short hairpin RNA (shRNA) in a MUC4-overexpressing (CD18/HPAF) pancreatic cancer cell line. The functional association of MUC4 expression with cancer phenotype was determined in various in vitro and in vivo studies. Our results show that the overexpression of MUC4 is associated with increased tumor cell proliferation, survival, invasiveness, and metastasis. Moreover, MUC4 interferes with the interaction between tumor cell and extracellular matrix (ECM) proteins, in part, by blocking the accessibility of integrins to ECM ligands. Furthermore, an altered expression of growth- and metastasis-associated genes is also reported on MUC4 down-regulation, indicating a novel role for MUC4 in cancer cell signaling.

Results

CD18/HPAF cells were transduced with the viral supernatant carrying either the MUC4 shRNA or the scrambled shRNA expression plasmids for effective silencing of MUC4 or to serve as control, respectively. The stable clones were selected in medium containing puromycin (3.0 μg/mL) and expanded, and the expression of MUC4 was examined by immunoblotting. All the clones (n = 19) selected from MUC4 short interfering RNA (siRNA)-transfected CD18/HPAF cells showed a 20% to 90% down-regulation of MUC4 (data not shown). Three clones (clones 9, 10, and 12) that consistently showed >70% reduced expression of MUC4 were pooled (CD18/HPAF-siMUC4) to avoid clonal variation in further investigations. The stable silencing of MUC4 in CD18/HPAF-siMUC4 cells compared with mixed population of scrambled shRNA-transfected cells (CD18/HPAF-Scr) was monitored by immunoblot and confocal microscopic analyses (Fig. 1A and B
). Western blot analysis showed ∼80% (on an average) down-regulation of MUC4 expression in CD18/HPAF-siMUC4 compared with control cells. Results of the confocal immunofluorescence microscopy were also consistent with the Western blot analysis, further confirming the reduced cellular expression of MUC4 in CD18/HPAF-siMUC4 cells compared with control cells. Additionally, we also examined the expression of MUC4 at transcript level by real-time reverse transcription-PCR and the results were in support with that obtained by immunoblot analysis (Supplementary Fig. S1).

Analyses of MUC4 expression in CD18/HPAF and its derived sublines: CD18/HPAF-Scr (mixed population of scrambled shRNA-transfected cells) and CD18/HPAF-siMUC4 (pooled population of MUC4 shRNA-transfected cells). A. Western blot analysis; a total of 10 μg protein from cell extracts was resolved by electrophoresis on a 2% SDS-agarose (for MUC4) and 10% SDS-polyacrylamide gel (for β-actin), transferred to polyvinylidene difluoride membrane, and incubated with anti-MUC4 or anti-β-actin monoclonal antibody. The membrane was then probed with horseradish peroxidase–labeled goat anti-mouse immunoglobulin, and the signal was detected using an electrochemiluminescence reagent kit. The intensity of signals was quantified by densitometry. β-Actin was used as an internal control. Columns, mean normalized fold difference in MUC4 protein expression levels (n = 3); bars, SE. *, P < 0.05. B. Expression analysis of MUC4 using confocal microscopy. CD18/HPAF, CD18/HPAF-Scr, and CD18/HPAF-siMUC4 cells were grown at low density on sterilized coverslips, washed, and fixed in ice-cold methanol at −20°C. The cells were blocked in 10% goat serum and incubated with the anti-MUC4 mouse monoclonal antibody. After washing, the cells were incubated with FITC-conjugated goat anti-mouse IgG. Cells were mounted on glass slides in antifade Vectashield mounting medium before observation under a Zeiss confocal laser-scanning microscope. Magnification, ×630. PI, propidium iodide.

The derived heterogeneous populations were studied to observe the effect of MUC4 silencing on tumor cell growth and metastasis in immunodeficient mice. All the mice injected orthotopically with the CD18/HPAF-Scr or CD18/HPAF-siMUC4 cells developed primary tumors. The tumor weight and volume were significantly lower (P = 0.02 and P < 0.001, respectively) in mice injected with the MUC4-knockdown (CD18/HPAF-siMUC4) cells compared with those injected with control (CD18/HPAF-Scr) cells. The mean weight and volume were 1,390.07 mg and 0.986 cm3 for the tumors developed from CD18/HPAF-Scr cells and 864.62 mg and 0.437 cm3 for those produced from the CD18/HPAF-siMUC4 cells, respectively (Supplementary Fig. S2A and B). The incidence of metastases to distant organ sites was also reported (Supplementary Table S1). All the mice injected with the CD18/HPAF-Scr cells developed metastases to one or multiple sites, whereas only 29% (4 of 14) of the mice injected with the MUC4-knockdown cells had detectable metastases. The most common sites of metastases were the lymph nodes, liver, and lungs. Specifically, the incidence of lymph node and liver metastases was lower in the mice injected with the MUC4-knockdown cells compared with those injected with the control cells (Supplementary Table S1). Immunohistochemical analysis of the primary tumor tissue sections was done with the anti-MUC4 mouse monoclonal antibody to monitor the expression of MUC4. Staining confirmed the down-regulated expression of MUC4 in tumors developed from CD18/HPAF-siMUC4 cells (Supplementary Fig. S3).

The siRNA-induced silencing of MUC4 in CD18/HPAF pancreatic cancer cells led to the suppression of both the tumor growth and metastasis. These results were consistent with our previous observation in which MUC4 expression was down-regulated by antisense technology (11). A reduced growth rate was also observed in the MUC4-knockdown cells in vitro (data not shown). Because the overall rate of cell growth is determined by the balance between cell proliferation and apoptosis, we next determined the proliferation and apoptotic indices of the CD18/HPAF-Scr and CD18/HPAF-siMUC4 pancreatic cancer cells. The proliferation index was determined by first synchronizing the cells at the G1-S boundary using a double thymidine block followed by propidium iodide staining and flow cytometry (Fig. 2
). The total percentage of CD18/HPAF-Scr cells that entered into the S phase was 42.51% compared with 17.71% in CD18/HPAF-siMUC4 cells, indicating a higher rate of proliferation in the MUC4-overexpressing (CD18/HPAF-Scr) cells.

Cell cycle analysis of MUC4-knockdown cells compared with control cells. CD18/HPAF cells, either knocked down for MUC4 expression or transfected with scrambled shRNA expression construct, were synchronized with double thymidine block. Following synchronization, cells were stained with propidium iodide and analyzed by fluorescence-activated cell sorting to evaluate the number of cells in different stages of cell cycles.

To analyze the apoptotic index, the MUC4-overexpressing and MUC4-silenced cells were serum starved and the extent of apoptosis and necrosis was determined by Annexin V and propidium iodide staining, respectively, followed by flow cytometry. The apoptotic index in CD18/HPAF-siMUC4 cells was nearly double to that reported for CD18/HPAF-Scr cells, whereas no changes in the necrosis were reported (Fig. 3
). Taken together, our data show that MUC4 enhances cell proliferation and favors the cell survival in CD18/HPAF pancreatic cancer cells.

Analysis of apoptotic index of MUC4-knockdown cells compared with control cells. CD18/HPAF cells, either knocked down for MUC4 expression or transfected with scrambled shRNA expression construct, were serum starved to induce apoptosis. The percentage of cells undergoing apoptosis or necrosis was measured by Annexin V and propidium iodide staining, respectively, followed by fluorescence-activated cell sorting analysis. Bottom left quadrant, being negative for both Annexin V and propidium iodide, shows the live cells; bottom right quadrant, being Annexin V positive and propidium iodide negative, shows the early apoptotic cells; top right quadrant, being both propidium iodide positive and Annexin V positive, shows the late apoptotic or necrotic cells. Columns, mean percentage of the apoptotic and necrotic cells (n = 3). *, P < 0.05.

The aggressiveness of a malignant cell is determined by its potential to invade the ECM and metastasize to distant sites. Several studies have supported the idea that the invasive and metastatic potential of cancer cells is intimately related to their motility (22). As the MUC4-overexpressing cells were more metastatic than the MUC4-knockdown cells, we examined if the expression of MUC4 is associated with enhanced motility of the CD18/HPAF pancreatic cancer cells. Consistent to our previous observation (11), MUC4-knockdown (CD18/HPAF-siMUC4) cells showed a significant decrease (P < 0.001) in motility (∼5-fold) compared with the control (CD18/HPAF-Scr) cells (Fig. 4A
). To further examine if the increased cell motility also correlated with the invasive potential of pancreatic cancer cells, we did an in vitro Matrigel invasion assay. We observed that nearly one third of the MUC4-knockdown (CD18/HPAF-siMUC4) cells invaded the Matrigel when compared with control (CD18/HPAF-Scr) cells (Fig. 4B).

Effect of MUC4 down-regulation on cell motility and invasion. Cells were plated on noncoated or Matrigel-coated membranes for motility (A) and invasion (B) assays, respectively, and incubated for 24 h. Medium containing 10% fetal bovine serum in the lower chamber was used as a chemoattractant. The cells that did not migrate through the Matrigel and/or pores in the membrane were removed, and cells on the other side of the membrane were stained and photographed under bright-field microscopy. Magnification, ×100. The number of cells migrated through the membrane was determined by averaging 10 random fields of view. Data are the number of cells per field of view. Columns, average of three independent experiments; bars, SE. *, P < 0.05. C. Actin organization. The MUC4-knockdown and control cells were grown on coverslips, fixed with 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and stained with Alexa Fluor 488–conjugated phalloidin. Increased lamellipodial structures were observed in parental and control-transfected CD18/HPAF cells compared with the MUC4-knockdown cells.

Given the key role played by the actin in defining cell shape and in orchestrating events related to cell movement (23), we next chose to focus our attention on the effect of MUC4 expression in reorganization of the actin cytoskeleton. In a highly motile cell, the actin filaments are organized on the mobile protruding edges, called lamellipodia, and can be visualized by confocal microscopy after staining with fluorescent dye-tagged phalloidin. Phalloidin is a fungal toxin that binds to filamentous actin and prevents its depolymerization into G-actin monomers (24). The staining of the control (CD18/HPAF-Scr) and MUC4-knockdown (CD18/HPAF-siMUC4) cells with Alexa Fluor 488–conjugated phalloidin showed the presence of many lamellipodial structures in the control cells, although they were less obvious in the MUC4 down-regulated cells (Fig. 4C). This suggests that the expression of MUC4 induces actin reorganization, thereby facilitating the motility and, in turn, the invasiveness of pancreatic cancer cells.

MUC4 Alters the Interaction of Pancreatic Tumor Cells with the ECM Components, in Part, by Interfering with the Accessibility of Integrins

Another important role envisaged for MUC4 is that of a modulator of cell-cell and cell-ECM interactions (16). In a previous study, we showed that MUC4 altered the cell-cell aggregation and cell adhesion on a plastic surface (11). To further study the role played by MUC4 in tumor cell-ECM interactions, CD18/HPAF-Scr and CD18/HPAF-siMUC4 cells were compared for their binding affinity to various ECM components (laminin I, collagen I, collagen IV, fibronectin, and basement matrix complex). The MUC4-knockdown cells showed significantly higher (P < 0.05) binding to laminin, basement matrix complex, collagen IV, collagen I, and fibronectin compared with the CD18/HPAF-Scr cells (Fig. 5
). These results suggest that the overexpression of MUC4 in pancreatic tumors reduces the ability of tumor cell to interact with ECM proteins.

Cell adhesion assay. Cells (25 × 103) were seeded in 96-well plate coated separately with laminin, collagen I, fibronectin, collagen IV, and basement membrane protein complex (BMC) proteins. Wells coated with bovine serum albumin and poly-l-lysine served as negative and positive controls, respectively. The cells were allowed to adhere for 1 h at 37°C, washed twice with PBS, and labeled with calcein-AM dye for 1 h at 37°C. The fluorescence of adhered cells was measured at an emission wavelength of 520 nm after excitation at 485 nm. The fluorescent intensity obtained in the negative control (bovine serum albumin–coated wells) was subtracted from the values obtained for different treatments (ECM component-coated wells). After that, a relative fluorescence unit (RFU) was calculated for all treatments with respect to the intensity value obtained with the positive control (poly-l-lysine–coated plates; n = 3). *, P < 0.05.

Integrins are the cell surface receptors for the ECM proteins (25). Given the observation that overexpression of MUC4 decreases adherence of tumor cell to ECM components, we decided to investigate the possible relationship between decreased adhesiveness and integrin expression in MUC4-overexpressing versus MUC4-knockdown cells. CD18/HPAF-Scr and CD18/HPAF-siMUC4 cells were seeded on anti-integrin-coated (α2, α3, and α5) plates and allowed to adhere for 1.0 h. A significantly reduced adhesion (P < 0.05) of the CD18/HPAF-Scr cells to the anti-integrin-coated plates was observed compared with the CD18/HPAF-siMUC4 cells (Fig. 6A
). Analysis of α2, α3, and α5 integrins by immunoblot assay, however, did not reveal any changes in their expression between MUC4-overexpressing and MUC4-knockdown cells (data not shown), which suggested a direct involvement of MUC4 in interfering with the accessibility of integrins to their coated antibodies. To obtain the evidence that cell surface expression of MUC4 inhibits the integrin-mediated adhesion, the cells were incubated with FITC-labeled monoclonal antibody (8G7) raised against the tandem repeat peptide of MUC4. Due to repetitive nature of epitope, the antibody binding leads to the clustering of the cell surface MUC4 molecules in multiple patches or in the form of a cap (Fig. 6B). The MUC4 clustering at the cell surface enhanced the adhesion of CD18/HPAF-Scr cells on the anti-integrin antibody-coated plates, although it had no significant effect on the binding of CD18/HPAF-siMUC4 cells (Fig. 6C). Furthermore, no changes in the adhesion were observed when cells were incubated with a nonspecific antibody (keyhole limpet hemocyanin antibody; data not shown). These observations suggest that MUC4 interferes with the accessibility of integrins to its ligands by causing the steric hindrance at the cell surface.

Cell surface integrin detection assay. A. Cell adhesion to anti-integrin-coated plates. Cells (25 × 103) were seeded in 96-well plate coated with anti-α2, anti-α3, and anti-α5 integrin antibodies. Wells coated with bovine serum albumin served as a negative control. Adhered cells were labeled with calcein-AM and fluorescence was measured at 520 nm after excitation at 485 nm (n = 3). *, P < 0.05. B. MUC4 capping at the cell surface. CD18/HPAF cells in suspension were incubated with FITC-conjugated anti-MUC4 antibody for 1 h at 4°C and washed twice with PBS-Tween 20. After washing, the cells were seeded on coverslips for 1 h at 37°C, fixed in methanol, and mounted on glass slides in antifade mounting medium. Immunostaining was observed under a Zeiss confocal laser-scanning microscope, and representative photographs were captured digitally using 510 LSM software. Magnification, ×100. C. Restoration of cell binding to anti-integrin-coated plates after MUC4 capping. MUC4 at the cell surface was capped in CD18/HPAF-Scr and CD18/HPAF-siMUC4 cells as in B. After washing, the cells were plated on 96-well plate coated with anti-α2, anti-α3, and anti-α5 integrin antibodies. Wells coated with bovine serum albumin served as a negative control. Adhered cells were labeled with calcein-AM, and fluorescence was measured at 520 nm after excitation at 485 nm (n = 3). *, P < 0.05.

MUC4 Enhances the Expression of Growth- and Metastasis-Associated Genes

DNA oligonucleotide microarrays representing ∼12,000 genes were used to identify genes regulated by MUC4 and potentially responsible for differences in the metastatic properties of MUC4-knockdown cells. The mRNA expression profile of CD18/HPAF-siMUC4 cells was compared with that of CD18/HPAF-Scr cells. A total of 173 genes was found to be differentially expressed in cells where MUC4 expression had been silenced. Of these, a few selected genes that were down-regulated or overexpressed in the MUC4-knockdown cells are listed in Tables 1
and 2
, respectively. Analysis of the data revealed that several growth- and metastasis-associated genes were down-regulated in the MUC4-knockdown cells. Of particular importance were the genes encoding LI-cadherin, CEACAM6, S100A4, tumor-associated calcium signal transducer-2, macrophage inhibitory cytokine 1, AnnexinA1, RAC1, epiregulin, and neuregulin-2 (Table 1). Among the genes that were up-regulated in MUC4-knockdown cells were those encoding for somatostatin, seladin-1, TP53, TP53BP, caspase-2, caspase-3, caspase-7, desmoglein-2, plakoglobin, and SMAC1. To validate our microarray data, expression of few differentially expressed genes was examined by Western blot analysis (Fig. 7
). The results of the Western blot analysis were in complete agreement with the microarray data, indicating that these could be functionally implicated in mediating the MUC4- modulated metastatic pathways.

Western blot analyses of selected differentially expressed genes (LI-cadherin, TP53, AnnexinA1, and plakoglobin) identified in microarray. A total of 30 μg protein from cell extracts was resolved by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and probed with respective antibodies. β-actin was used as an internal control. The expression of these genes was in accordance with the microarray data.

Discussion

Our previous studies have implicated MUC4 mucin as an important player in the pathogenesis of pancreatic cancer (6, 7, 11, 12). In the present work, we provide additional evidence to buttress our earlier findings and provide mechanistic basis for the role of MUC4 in pancreatic cancer progression. We show here that siRNA-mediated silencing of MUC4 restrains growth and metastasis of CD18/HPAF pancreatic adenocarcinoma cells. Our in vitro studies show that the increase in tumor cell growth associated with MUC4 overexpression results from enhanced proliferation and reduced cell death. MUC4 expression is also associated with actin reorganization and cancer cell invasiveness. Moreover, the presence of MUC4 on the cell surface interferes with the tumor cell-ECM interaction, in part, by inhibiting integrin-mediated cell adhesion. Further, MUC4 down-regulation was found to alter the expression of several growth- and metastasis-associated genes.

Normal cell growth is maintained by the balance between cell proliferation and cell death. While studying the cause for the reduced tumor growth in MUC4-knockdown cells, we observed that an increase in MUC4 expression is associated with a higher rate of cell proliferation and reduced apoptosis (Figs. 2 and 3). In a previous study, the overexpression of SMC/Muc4 (the rat homologue of MUC4) was reported to accelerate the growth of xenotransplanted A375 melanoma cells in host animals by suppressing the apoptosis (18). Muc4 was shown to act as an intramembrane ligand for ErbB2/HER2/neu, inducing its limited phosphorylation. It was also suggested that Muc4-induced ErbB2/neu signaling might mediate the antiapoptotic function of Muc4 (18). Consistent with our previous report (11), we also observed a down-regulation of the receptor tyrosine kinase, HER2, and its downstream signaling in MUC4-knockdown pancreatic cancer cells (data not shown). A down-regulation of growth-promoting genes and an up-regulation of apoptosis-inducing genes were also observed in CD18/HPAF-siMUC4 cells (Tables 1 and 2). This may explain the changes in the pancreatic cancer cell growth in response to MUC4 silencing. Particularly, MUC4-mediated down-regulation of tumor suppressors, such as TP53 and TP53BP1, might aid the MUC4-expressing tumor cells in surpassing the cell cycle checkpoints and, thus, facilitate their uncontrolled cell growth (26). Moreover, the down-regulation of apoptosis mediators (caspase-2, caspase-3, caspase-7, TP53I11, and SMAC; refs. 27-29) in MUC4-overexpressing control cells might lead to enhanced cell survival.

Apart from increased tumorigenicity, MUC4-expressing cells also showed an enhanced tendency to metastasize to distant organs. The process of metastasis is a complex phenomenon regulated by many components, which work in tandem to facilitate the detachment of tumor cells from their site of origin and subsequent spread to secondary sites (30). Similar to our previous observations (11), the MUC4 down-regulated cells showed a significant decrease in motility (Fig. 4). Furthermore, our present study showed that MUC4-knockdown cells were less invasive compared with the control cells (Fig. 4B). Cell motility and invasiveness are typically associated with the actin reorganization (23). In a highly motile cell, the actin is organized on the mobile protruding edges called lamellipodia (23). An increase in lamellipodia-like structures was observed in the MUC4-expressing cells, although these structures were less obvious in the knockdown cells (Fig. 4C). Thus, our findings lend support to the novel idea that MUC4 induces remodeling of the actin cytoskeleton and thereby contributes to enhanced cell motility and invasiveness. The molecular basis of MUC4-induced changes in actin organization is yet to be investigated. Cell-cell interactions, including cadherin-mediated interactions, restrict cell motility (31). Steric hindrance caused by the large extracellular domain of MUC4 may disrupt these interactions (11) and might indirectly be responsible for rearrangements in the actin cytoskeleton and, hence, enhanced motility. Alternatively, as discussed later, MUC4 expression might affect multiple signaling pathways either by affecting the ligand accessibility to their corresponding receptors or by directly interacting with the signal transducers, leading to transcriptional or posttranscriptional activation of the regulators of cell motility. Therefore, it can be suggested that MUC4 influences the cancer cell signaling in favor of the metastatic behavior of the tumor cells.

Another important finding of this study was the enhanced tumor cell-ECM interaction on abrogation of MUC4 expression (Fig. 5). An antiadhesive property of transmembrane mucins is predicted due to the presence of a highly glycosylated tandem repeat domain, which may sterically hinder their homotypic and heterotypic interactions (1). Our results showed that adhesion of pancreatic tumor cells to laminin, collagen IV, collagen I, and fibronectin and basement membrane proteins was significantly increased in MUC4 down-regulated cells (Fig. 5). The interaction of the cell with the ECM components is mediated by a family of cell surface receptors, integrins. Till date, more than 20 different integrins and several additional splice variants have been identified, with a specific subset being expressed by each cell type (25, 32). MUC4 masked the surface epitopes of the integrins (α2, α3, and α5) to their respective antibodies (Fig. 6), and hence, it can be suggested that MUC4 interferes with the integrin-mediated cell adhesion by restricting their accessibility to the ligands. A similar observation has also been reported for SMC/Muc4 (16). Transfection of A375 human melanoma cells with SMC resulted in a significant reduction of cell-cell and cell-matrix interactions. Furthermore, the extent of interference was directly correlated with the size of the tandem repeat domain (16). Similar results have been documented for MUC1 by overexpressing it in transformed epithelial and cancer cell lines (33).

An altered expression of genes is observed in response to MUC4 down-regulation (Tables 1 and 2). Among the various growth- and metastasis-associated genes up-regulated in MUC4- overexpressing cells are LI-cadherin, CEACAM6, tumor-associated calcium signal transducer-2, AnnexinA1, thrombomodulin, epiregulin, S100A4, and macrophage inhibitory cytokine 1 (34-36). An up-regulation of LI-cadherin has been shown to correlate with lymph node metastasis in gastric cancer (37, 38). Lymph node is one of the most metastasized sites in pancreatic cancer. CEACAM6 expression is correlated with a poor survival in pancreatic cancer patients (39). Furthermore, silencing of CEACAM6 expression by using siRNA in pancreatic adenocarcinoma cells suppresses anoikis resistance in vitro and metastatic ability in vivo (40). An overexpression of AnnexinA1, a Ca2+-dependent phospholipid binding protein, in pancreatic adenocarcinoma is correlated with differentiation of cancer cells during tumorigenesis (41). It has previously been identified as being significantly up-regulated in pancreatic head adenocarcinomas by cDNA microarray analysis (42). MUC4 also up-regulated the expression of Dynein, a protein associated with the retrograde organelle transport and some aspects of mitosis, and Sec61, which is associated with protein transport (43). An up-regulation of the NCK-associated protein 1, RAC1, ARP2/3 protein complex subunit p21 (ARC21), and ARP1 was also reported in the MUC4-overexpressing cells when compared with the MUC4-knockdown cells. All these proteins are key regulators of cell motility (44), which was significantly higher in MUC4-overexpressing cells compared with MUC4-knockdown cells. RAC1 and NCK are known to stimulate DNA synthesis in the presence of fibroblast growth factor-2 via activation of c-Jun NH2-terminal kinase pathway (45). Hence, the RAC1 and NCK protein networks might be indirectly responsible for the MUC4-mediated induction of cell proliferation.

MUC4-associated changes in gene expression indicate its important role in cell signaling. Transmembrane mucins are hypothesized to serve as sensors of the external environment either through extracellular domain-mediated ligand binding or as a consequence of altered conformations that result from changes in the external biochemical conditions (pH, ionic composition, and physical interactions; refs. 1, 46). In addition, they can transduce signals via the posttranslational modifications of their cytoplasmic tails (1, 46). Cytoplasmic tail of MUC1 is phosphorylated by various kinases, including epidermal growth factor receptor, c-src, glycogen synthase kinase-3β, protein kinase C-δ, Lyn, Lck, and Zap-70, in response to different stimuli. Phosphorylated MUC1 cytoplasmic tail activates different intracellular signaling cascades that can trigger β-catenin-TCF/LEF–dependent, TP53-dependent, or estrogen receptor-α–dependent transcriptional activity and, hence, facilitate tumor progression (47-49). MUC4 has a short cytoplasmic tail (composed of 22 amino acids) that may be engaged in signal transduction and therefore warrants future investigation. Nonetheless, MUC4 may also act as a modulator of cancer cell signaling via disrupting the normal protein-protein interactions and by forming novel, cancer-associated interactions.

In conclusion, our data provide novel evidence supporting the role of MUC4 mucin in the progression of pancreatic cancer. We show that MUC4 sterically regulates the accessibility of cell surface receptors to their ligands. Further, we also show that MUC4 is associated with enhanced cell invasion, altered tumor cell-ECM interaction, and a change in expression of various growth- and metastasis-associated genes. Hence, we propose that MUC4 can be a useful target in the development of novel therapeutic strategies for the treatment of pancreatic cancer.

Materials and Methods

Construction of Plasmid Expressing shRNA against MUC4 and Retroviral Generation

The MUC4-specific siRNAs (19 nucleotides) were designed and tested for effective MUC4 silencing in transient assays. One of the MUC4 siRNAs (5′-CAGCGACACTAGAGGGACA-3′) located 151 bp downstream of ATG was highly efficient and therefore used for generating a stable shRNA-expressing construct in the pSUPER-retro-puro vector according to the manufacturer's instructions. In brief, two 64-mer oligonucleotides (forward and reverse) containing the 19-bp target sequence in sense and antisense orientation on both sides of the linker TTCAAGAGA were designed. The BglII and HindIII restriction sites were located at 5′ and 3′ regions of 64-mers, respectively, to allow direct ligation of the annealed MUC4 double-strand primer into the pSUPER-retro-puro vector. The annealed double-stranded oligonucleotides were phosphorylated using polynucleotide kinase (Roche Diagnostics, Mannheim, Germany) and ligated into the digested pSUPER-retro-puro vector. Successful cloning was ascertained by restriction digestion and sequencing. Ecotropic phoenix packaging cells (gift from Dr. Parmender Mehta, University of Nebraska Medical Center, Omaha, NE) were transfected with the pSUPER-retro-puro vector containing either the MUC4 shRNA insert (pSUPER-retro-puro.siMUC4) or a scrambled sequence (pSUPER-retro-puro.Scr) using LipofectAMINE (Invitrogen, San Diego, CA) following the manufacturer's protocol. Media containing infection-competent retroviruses were collected 48 h after transfection. Polybrene (4 μg/mL) was used to augment the infection efficiency.

Cell Culture and Transfection

CD18/HPAF pancreatic cancer cells were cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics (100 μg/mL of penicillin and streptomycin) at 37°C with 5% CO2 in a humidified atmosphere. Retroviral supernatants from Phoenix cultures transfected with the pSUPER-retro-puro.siMUC4 or pSUPER-retro-puro.Scr were used for stable transduction of CD18/HPAF cells. Stable clones were then selected in medium containing puromycin (3 μg/mL; InvivoGen, San Diego, CA). The puromycin-resistant colonies were isolated by the ring cloning method and maintained in medium supplemented with puromycin. Medium was replaced with complete medium without antibiotic supplement at least 5 days before any analysis.

Immunoblot and Confocal Immunofluorescence Microscopic Analyses

The CD18/HPAF and derived cell lines were processed for Western blotting and confocal microscopy as described previously (50). In brief, a total of 10 to 30 μg of protein from the cell extracts was resolved by electrophoresis on either a 2.0% SDS-agarose gel (for MUC4) or a 10% SDS-polyacrylamide gel (for other proteins) and transferred to polyvinylidene difluoride membrane. The membrane was probed with anti-MUC4 (1:1,000), anti-TP53 (1:1,000; gift from Dr. Xu Luo, University of Nebraska Medical Center), anti-LI-cadherin (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-AnnexinA1 and anti-plakoglobin (1:100; gift from Dr. Keith Johnson, University of Nebraska Medical Center), and anti-β-actin (1:10,000; Sigma, St. Louis, MO) antibodies. The membrane was then incubated with horseradish peroxidase–labeled appropriate secondary immunoglobulin, and the signal was detected using an electrochemiluminescence reagent kit (Amersham Pharmacia, Piscataway, NJ).

For immunofluorescence labeling, cells were grown at low density on sterilized coverslips. After washing with 0.1 mol/L HEPES containing Hanks' buffer, the cells were fixed in ice-cold methanol at −20°C for 2 min. Nonspecific blocking was done with 10% goat serum containing 0.05% Tween 20 for at least 30 min followed by incubation with the anti-MUC4 monoclonal antibody (8G7) in PBS for 90 min at room temperature. Cells were then washed and incubated with FITC-conjugated goat anti-mouse secondary antibodies for 60 min. After washing, the coverslips were mounted on glass slides in antifade Vectashield mounting medium (Vector Laboratories, Burlingame, CA). For actin filament staining, the cells were grown on glass coverslips and fixed with 4% formaldehyde in PBS for 10 min at room temperature. The fixed cells were washed with PBS and permeabilized with 0.2% Triton X-100 in PBS for 5 min. After washing, the cells were stained with Alexa Fluor 488-phalloidin (Molecular Probes, Eugene, OR) for 20 min at room temperature. Cells were washed twice with PBS-Tween 20 and mounted on glass slides in antifade Vectashield mounting medium. Immunostaining was observed under a Zeiss (Carl Zeiss Microimaging, Thornwood, NY) confocal laser-scanning microscope, and representative photographs were captured digitally using the 510 LSM software.

Cell Cycle Analysis

The cells were first synchronized at the G1-S stage using a double thymidine block. In brief, the cells were treated with thymidine (Sigma) at 2 mmol/L final concentration for 18 h and washed twice with PBS. The cells were then incubated with fresh medium without thymidine for 9 h at 37°C. The thymidine was added again to a final concentration of 2 mmol/L and incubated for another 18 h and washed with PBS, and fresh medium was added. Following synchronization, the cells were stained with 1 mL propidium iodide (1 μg/mL) and analyzed by flow cytometry.

Apoptosis Assay

Apoptosis was measured by using the Annexin V-FITC apoptosis detection kit (Roche Diagnostics, Indianapolis, IN). The cells were grown in serum-free medium for 72 h. Apoptosis was detected by staining the cells with Annexin V and propidium iodide solution followed by flow cytometry.

Cell Motility Assay

Cells (1 × 106) were plated in the top chamber of noncoated polyethylene terephthalate membranes (six-well insert, pore size of 8 μm; Becton Dickinson, Franklin Lakes, NJ). The bottom chamber contained 1.0 mL DMEM supplemented with 10% fetal bovine serum. The cells were incubated for 24 h at 37°C, and the cells that did not migrate through the pores in the membrane were removed by wiping the membrane with a cotton swab. Cells that passed through the membrane pores were stained with a Diff-Quick cell staining kit (Dade Behring, Inc., Newark, DE). Cells in 10 random fields of view at ×100 magnification were counted and expressed as the average number of cells per field of view. Three independent experiments were done in each case. The data were represented as the average of the three independent experiments.

Cell Invasion Assay

Cells (1 × 105) were seeded on Matrigel-coated membrane inserts (BD Biosciences, Bedford, MA). The bottom chamber contained 0.75 mL DMEM supplemented with 10% fetal bovine serum as a chemoattractant. After incubation for 24 h at 37°C, the cells that remained inside the insert were removed with a cotton swab, and cells that had penetrated the Matrigel to invade to the lower surface of the membrane were fixed in methanol and stained using a Diff-Quick reagent kit. After air drying the membrane, the cells were counted at a magnification of ×100 in 10 random fields of view under a microscope. Assays were done thrice in triplicate.

Cell Adhesion Assay

Cells were harvested and resuspended at a density of 2.5 × 105/mL. A total of 100 μL of the cell suspension (25 × 103 cells) was seeded in triplicate onto the laminin, collagen, fibronectin, and basement membrane complex protein-coated 96-well plates (Calbiochem, La Jolla, CA) and incubated for 1 h at 37°C. After incubation, the cell suspension was discarded and the wells were gently washed twice with PBS. The cells that adhered to the wells were incubated with 100 μL of calcein-AM dye for 1 h at 37°C. The fluorescence of the samples was measured using the fluorescence plate reader at an excitation wavelength of 485 nm and emission wavelength of 520 nm.

Cell Surface Integrin Detection Assay

For the integrin detection assay, goat anti-mouse IgG-coated 96-well plates were incubated with 100 μL of anti-α2, anti-α3, and anti-α5 integrin antibodies (Calbiochem) in separate wells and incubated for 30 min at 37°C. Following incubation, the unbound antibodies were washed off. Cells (25 × 103) were seeded in triplicate in anti-α2, anti-α3, and anti-α5 antibody captured plates and incubated for 1 h at 37°C. After incubation, the cell suspension was discarded and the wells were gently washed twice with PBS. The adhered cells were incubated with 100 μL of calcein-AM dye for 1 h at 37°C. The fluorescence of the samples was measured using the fluorescence plate reader at an excitation wavelength of 485 nm and emission wavelength of 520 nm.

Cell Surface Capping of MUC4

CD18/HPAF cells in suspension were incubated with FITC-conjugated anti-MUC4 antibody (25 μg/mL) for 1 h at 4°C. After washing, the cells were plated on coverslips and incubated for 75 min at 37°C for adherence and fixed in methanol. These cells were then washed twice with PBS-Tween 20 and mounted on glass slides in antifade Vectashield mounting medium. Immunostaining was observed under a Zeiss confocal laser-scanning microscope, and representative photographs were captured digitally.

cDNA Microarray Hybridization and Analysis

RNA (60 μg) from CD18/HPAF-Scr and CD18/HPAF-siMUC4 cell line was fluorescently labeled with Cy3- and Cy5-conjugated dCTP (Amersham Pharmacia) using anchored oligo(dT) primers and SuperScript II reverse transcriptase (Life Technologies, Gaithersburg, MD). After probe generation, residual RNA was hydrolyzed by treatment with NaOH and the unincorporated nucleotides were removed by a MicroCon YM-30 column (Amicon, Billerica, MA). The latter step also served to concentrate the labeled cDNAs. Cy3- and Cy5-labeled cDNAs were combined and diluted to 60 μL with 3.5× SSC/0.15% SDS hybridization solution. To reduce nonspecific hybridization, the hybridization solution also contained 10 μg of poly(deoxyadenylate) (Amersham Pharmacia), 2.5 μg of yeast tRNA (Sigma), and 12.5 μg of human Cot1 DNA (Boehringer-Mannheim, Indianapolis, IN). The fluorescent probes were denatured and hybridized to glass slides featuring ∼12,000 oligonucleotides overnight at 65°C. Slides were washed successively in 1× SSC/0.1% SDS, 1× SSC, and 0.2× SSC for 2 min each to remove excess probe. The microarrays were dried by centrifugation for 5 min at 1,000 rpm and scanned immediately with a ScanArray 4000 confocal laser system (Perkin-Elmer, Wellesley, MA). Fluorescent intensity of hybridization signals for each spot was determined automatically and corrected for background. Calculated intensities correlate linearly with the concentration of mRNAs present in the total RNA population because the amount of cDNA attached to the membrane was in excess and the background hybridization signals were sufficiently low. For a quantitative difference in gene expression between arrays, the intensity value of each known gene was normalized in two ways: to the intensity values of the designed housekeeping genes and to the sum of the intensity values of all of the genes. We found no significant difference in the normalization coefficients generated by the two methods. Comparison of two cell lines (CD18/HPAF-Scr and CD18/HPAF-siMUC4) RNA population was done in two separate parallel hybridization experiments. Genes that showed an average induction or reduction of ≥2-fold in both hybridization experiments were considered to be differentially expressed. Correlations and differences in gene expression between CD18/HPAF-Scr and CD18/HPAF-siMUC4 cells were also compared by scatter plot analysis, in which each point represents a particular gene.

Statistical Analyses

Mean tumor weight and mean tumor volume were compared between groups using an independent sample t test. A P value of <0.05 was considered as statistically significant.

Footnotes

Grant support: NIH grant CA 78590.

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